Helaina Inc., 345 Park Avenue South, 5th Floor, New York, NY 10010, USA.
X-ray Crystallography Core Facility, The Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802, USA.
Analyst. 2024 Jun 24;149(13):3636-3650. doi: 10.1039/d4an00333k.
This work presents a thorough characterization of Helaina recombinant human lactoferrin (rhLF, Effera™) expressed in a yeast system at an industrial scale for the first time. Proteomic analysis confirmed that its amino acid sequence is identical to that of native human LF. N-linked glycans were detected at three known glycosylation sites, namely, Asparagines-156, -497, and -642 and they were predominantly oligomannose structures having five to nine mannoses. Helaina rhLF's protein secondary structure was nearly identical to that of human milk lactoferrin (hmLF), as revealed by microfluidic modulation spectroscopy. Results of small-angle X-ray scattering (SAXS) and analytical ultracentrifugation analyses confirmed that, like hmLF, Helaina rhLF displayed well-folded globular structures in solution. Reconstructed solvent envelopes of Helaina rhLF, obtained through the SAXS analysis, demonstrated a remarkable fit with the reported crystalline structure of iron-bound native hmLF. Differential scanning calorimetry investigations into the thermal stability of Helaina rhLF revealed two distinct denaturation temperatures at 68.7 ± 0.9 °C and 91.9 ± 0.5 °C, consistently mirroring denaturation temperatures observed for apo- and holo-hmLF. Overall, Helaina rhLF differed from hmLF in the N-glycans they possessed; nevertheless, the characterization results affirmed that Helaina rhLF was of high purity and exhibited globular structures closely akin to that of hmLF.
本工作首次全面表征了在酵母系统中以工业规模表达的重组人乳铁蛋白（rhLF，Effera™）。蛋白质组学分析证实其氨基酸序列与天然人 LF 完全相同。在三个已知糖基化位点（即天冬酰胺-156、-497 和-642）检测到 N-连接糖基化，它们主要是具有五到九个甘露糖的寡甘露糖结构。微流控调制光谱显示，Helaina rhLF 的蛋白质二级结构与人乳乳铁蛋白（hmLF）几乎相同。小角 X 射线散射（SAXS）和分析超速离心分析的结果证实，与 hmLF 一样，Helaina rhLF 在溶液中显示出良好折叠的球形结构。通过 SAXS 分析获得的 Helaina rhLF 溶剂包络的重建表明，与报道的铁结合天然 hmLF 的晶体结构非常吻合。对 Helaina rhLF 热稳定性的差示扫描量热法研究表明，在 68.7±0.9°C 和 91.9±0.5°C 处有两个明显的变性温度，与 apo-和 holo-hmLF 观察到的变性温度一致。总体而言，Helaina rhLF 与 hmLF 所具有的 N-聚糖不同；然而，表征结果证实 Helaina rhLF 具有高纯度，并表现出与 hmLF 非常相似的球形结构。
